Impact of chronic hyperglycemia on Small Heat Shock Proteins in diabetic rat brain.

Small heat shock proteins (sHsps) are a family of proteins. Some are induced in response to multiple stimuli and others are constitutively expressed. They are involved in fundamental cellular processes, including protein folding, apoptosis, and maintenance of cytoskeletal integrity. Hyperglycemia created during diabetes leads to neuronal derangements in the brain. In this study, we investigated the impact of chronic hyperglycemia on the expression of sHsps and heat shock transcription factors (HSFs), solubility and aggregation of sHsps and amyloidogenic proteins, and their role in neuronal apoptosis in a diabetic rat model. Diabetes was induced in Sprague-Dawley rats with streptozotocin and hyperglycemia was maintained for 16 weeks. Expressions of sHsps and HSFs were analyzed by qRT-PCR and immunoblotting in the cerebral cortex. Solubility of sHsps and amyloidogenic proteins, including α-synuclein and Tau, was analyzed by the detergent soluble assay. Neuronal cell death was analyzed by TUNEL staining and apoptotic markers. The interaction of sHsps with amyloidogenic proteins and Bax was assessed using co-immunoprecipitation. Hyperglycemia decreased Hsp27 and HSF1, and increased αBC, Hsp22, and HSF4 levels at transcript and protein levels. Diabetes induced the aggregation of αBC, Hsp22, α-synuclein, and pTau, as their levels were higher in the insoluble fraction. Additionally, diabetes impaired the interaction of αBC with α-synuclein and pTau. Furthermore, diabetes reduced the interaction of αBC with Bax, which may possibly contribute to neuronal apoptosis. Together, these results indicate that chronic hyperglycemia induces differential responses of sHsps by altering their expression, solubility, interaction, and roles in apoptosis.

Functional analysis of a novel orthologous small heat shock protein (shsp) hsp21.8a and seven species-specific shsps in Tribolium castaneum.

Small heat shock proteins (sHSPs) are important modulators of insect survival. Previous research revealed that there is only one orthologous cluster of shsps in insects. Here, we identified another novel orthologous cluster of shsps in insects by comparative analysis. Multiple stress experiments and function investigation of Tchsp21.8a belonging to this orthologous cluster and seven species-specific shsps were performed in the stored-grain pest Tribolium castaneum. The results indicated that expression of Tchsp21.8a showed weak responses to different stresses. However, expressions of most species-specific shsps exhibited hyper-responses to heat stress, and expressions of all species-specific shsps displayed diverse responses during other stresses to protect beetles in a cooperative manner. Additionally, Tchsp21.8a and species-specific Tcshsp19.7 played important roles in the development of T. castaneum, and all Tcshsps had a certain impact on the fecundity. Our work created a comprehensive reliable scaffold of insect shsps that can further provide instructive insights to pest bio-control.

Nucleopolyhedroviruses (NPV) induce the expression of small heat shock protein 25.4 in Antheraea pernyi.

Nucleopolyhedroviruses (NPVs) is one group of Baculoviruses. The infection of NPV in silkworm is often lethal. To investigate the effective measures to stop the infection of NPV, we cloned cDNA encoding small heat shock protein 25.4 in Antheraea pernyi (Ap-HSP25.4). The translated amino acid sequence consisted of 223 residues with a calculated molecular mass of 25.4kDa and an isoelectronic point (pI) of 4.93. Quantitative real-time PCR was used to investigate the expression patterns and distribution profiles of Ap-sHSP25.4 before and after challenged with NPV. We found that the inhibitors of eicosanoid synthesis could suppress the transcription of Ap-sHSP25.4 in the fat body in a dose dependent manner. And arachidonic acid induced the expression of Ap-sHSP25.4. Thus, we concluded that sHSPs may be promising candidates to boost insect immunity in practice.

Overexpression of Small Heat Shock Protein Enhances Heat- and Salt-Stress Tolerance of Bifidobacterium longum NCC2705.

Bifidobacteria are probiotics that are incorporated live into various dairy products. They confer health-promotive effects via gastrointestinal tract colonization. However, to provide their health-beneficial properties, they must battle the various abiotic stresses in that environment, such as bile salts, acids, oxygen, and heat. In this study, Bifidobacterium longum salt- and heat-stress tolerance was enhanced by homologous overexpression of a small heat shock protein (sHsp). A positive contribution of overproduced sHsp to abiotic stress tolerance was observed when the bacterium was exposed to heat and salt stresses. Significantly higher survival of B. l ongum NCC2705 overexpressing sHsp was observed at 30 and 60 min into heat (55 °C) and salt (5 M NaCl) treatment, respectively. Thermotolerance analysis at 47 °C with sampling every 2 h also revealed the great potential tolerance of the engineered strain. Cell density and acid production rate increased for the sHsp-overexpressing strain after 8 and 10 h of both heat and salt stresses. In addition, tolerance to bile salts, low pH (3.5) and low temperature (4 °C) was also increased by homologous overexpression of the sHsp hsp20 in B. l ongum. Results revealed that hsp20 overexpression in B longum NCC2705 plays a positive cross-protective role in upregulating abiotic responses, ensuring the organism's tolerance to various stress conditions; therefore, sHsp-overexpressing B. l ongum is advised for fermented dairy foods and other probiotic product applications.

Expression and induction of small heat shock proteins in rat heart under chronic hyperglycemic conditions.

The induction of small heat shock proteins (sHsp) is observed under various stress conditions to protect the cells and organisms from adverse events including diabetes. Diabetic cardiomyopathy is a common complication of diabetes. Therefore, in this study, we investigated the expression of sHsp under chronic hyperglycemic conditions in rat heart. Hyperglycemia was induced in WNIN rats by intraperitoneal injection of streptozotocin and maintained for a period of 12weeks. Expression of sHsp, phosphorylation and translocation of phosphoforms of Hsp27 and αB-crystallin (αBC) from cytosolic fraction to cytoskeletal fraction was analyzed. While the expression of MKBP, HspB3, αBC was found to be increased in diabetic heart, expression of Hsp20 was decreased. Chronic hyperglycemia further induced phosphorylation of αBC at S59, S45, Hsp27 at S82, p38MAPK and p44/42MAPK. However, pS59-αBC and pS82-Hsp27 were translocated from detergent-soluble to detergent-insoluble fraction under hyperglycemic conditions. Furthermore, the interaction of pS82-Hsp27 and pS59-αBC with desmin was increased under hyperglycemia. However, the interaction of αBC and pS59-αBC with Bax was impaired by chronic hyperglycemia. These results suggest up regulation of sHsp (MKBP, HspB3 and αBC), phosphorylation and translocation of Hsp27 and αBC to striated sarcomeres and impaired interaction of αBC and pS59-αBC with Bax under chronic hyperglycemia.

Expression analysis of nine small heat shock protein genes from Tamarix hispida in response to different abiotic stresses and abscisic acid treatment.

Heat shock proteins (HSPs) play important roles in protecting plants against environmental stresses. Furthermore, small heat shock proteins (sHSPs) are the most ubiquitous HSP subgroup with molecular weights ranging from 15 to 42 kDa. In this study, nine sHSP genes (designated as ThsHSP1-9) were cloned from Tamarix hispida. Their expression patterns in response to cold, heat shock, NaCl, PEG and abscisic acid (ABA) treatments were investigated in the roots and leaves of T. hispida by real-time RT-PCR analysis. The results showed that most of the nine ThsHSP genes were expressed at higher levels in roots than in leaves under normal growth condition. All of ThsHSP genes were highly induced under conditions of cold (4 °C) and different heat shocks (36, 40, 44, 48 and 52 °C). Under NaCl stress, all nine ThsHSPs genes were up-regulated at least one stress time-point in both roots and leaves. Under PEG and ABA treatments, the nine ThsHSPs showed various expression patterns, indicating a complex regulation pathway among these genes. This study represents an important basis for the elucidation of ThsHSP gene function and provides essential information that can be used for stress tolerance genetic engineering in future studies.

Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons.

The so-called stress response involving up-regulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia, or oxidative stress. Some members of the group of small Hsps (sHsps) seem to play a neuroprotective role in the brain. Here we analyzed the expression of all 11 sHsps in the rat brain by using RNA in situ hybridization and quantitative real-time RT-PCR. Additionally, we investigated sHsps in cultured neurons exposed to heat shock. We found seven sHsps to be expressed in the rat brain, with HspB5 (αB-crystallin), HspB6 (Hsp20), and HspB11 (Hsp16.2) showing the highest expression levels (4-24% of reference genes) followed by HspB1 (Hsp25) and HspB8 (Hsp22; 0.1-2% of reference genes), all being widely expressed in the brain areas investigated. HspB2 (MKBP) and HspB3, however, showed selective expression in only some regions (B2: cortex and hippocampus, B3: cortex and cerebellum). Whereas HspB5 was expressed mainly in the white matter, HspB6 showed the greatest expression in the cerebellar cortex, and HspB11 was widely distributed over the whole brain. In cultured hippocampal neurons, heat shock led to an increase of HspB1 and HspB8 mRNA and additionally HspB5 protein. Our data indicate that the sHsps induced by heat shock, HspB1, B5, and B8, might be especially involved in neuroprotection under stress conditions. The other sHsps showing constant neuronal expression may play a constitutive role or may be up-regulated and important in types of stresses other than heat shock.

Identification of the Phr-dependent heat shock regulon in the hyperthermophilic archaeon, Thermococcus kodakaraensis.

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a putative transcriptional regulator (Tk-Phr) that is orthologous to the Pyrococcus furiosus Phr (Pf-Phr). Pf-Phr, a transcriptional regulator, represses genes encoding the small heat shock protein (sHSP), AAA(+) ATPase and Pf-Phr itself under normal growth temperatures. Here we constructed a gene disruption strain of Tk-Phr (strain KHR1). KHR1 cells showed similar specific growth rates with those of the wild-type strain under various temperatures. A whole genome microarray analysis was performed between KHR1 and wild-type cells grown at 80 degrees C. Transcript levels of more than 20 genes were significantly higher in KHR1 cells. Most genes contained a sequence motif virtually identical to that of Pf-Phr in their 5'-flanking regions. The Tk-Phr regulon included genes encoding sHSP, AAA(+) ATPase, prefoldin, RecA superfamily ATPase and Tip49. On the other hand, more than half of the members in the regulon encoded conserved/hypothetical proteins, raising the possibility that these proteins participate in unidentified processes of the heat shock response. In contrast, Tk-Phr deletion did not lead to dramatic increase in transcript and protein levels of a chaperonin (CpkB) previously shown to respond to heat shock, suggesting the presence of a second, Phr-independent heat shock response mechanism in T. kodakaraensis.

Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a Gram-negative plant-pathogenic bacterium that causes crown gall disease by transferring and integrating its transferred DNA (T-DNA) into the host genome. We characterized the chromosomally encoded alpha-crystallin-type small heat-shock protein (alpha-Hsp) HspL, which was induced by the virulence (vir) gene inducer acetosyringone (AS). The transcription of hspL but not three other alpha-Hsp genes (hspC, hspAT1, hspAT2) was upregulated by AS. Further expression analysis in various vir mutants suggested that AS-induced hspL transcription is not directly activated by the VirG response regulator but rather depends on the expression of VirG-activated virB genes encoding components of the type IV secretion system (T4SS). Among the 11 virB genes encoded by the virB operon, HspL protein levels were reduced in strains with deletions of virB6, virB8 or virB11. VirB protein accumulation but not virB transcription levels were reduced in an hspL deletion mutant early after AS induction, implying that HspL may affect the stability of individual VirB proteins or of the T4S complex directly or indirectly. Tumorigenesis efficiency and the VirB/D4-mediated conjugal transfer of an IncQ plasmid RSF1010 derivative between A. tumefaciens strains were reduced in the absence of HspL. In conclusion, increased HspL abundance is triggered in response to certain VirB protein(s) and plays a role in optimal VirB protein accumulation, VirB/D4-mediated DNA transfer and tumorigenesis.

ArHsp22, a developmentally regulated small heat shock protein produced in diapause-destined Artemia embryos, is stress inducible in adults.

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors.

BACKGROUND: Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor. METHODS: Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given. Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting. RESULTS: Low grade (grades 1-2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples. CONCLUSION: Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.

Cloning and expression analysis of p26 gene in Artemia sinica.

The protein p26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. A 542 bp fragment of the p26 gene was cloned and sequenced. The fragment encoded 174 amino acid residues and the amino acid sequence contained the alpha-crystallin domain. Phylogenetic analysis showed that eight Artemia populations were divided into four major groups. Artemia sinica (YC) belonged to the East Asia bisexual group. Expression of the p26 gene at different developmental stages of A. sinica was quantified using real-time quantitative polymerase chain reaction followed by cloning and sequencing. The relationship between the quantity of p26 gene expression and embryonic development was analyzed. The results indicated that massive amounts of p26 were expressed during the development of A. sinica. At the developmental stage of 0 h, A. sinica expressed the highest level of p26. As development proceeded, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of 16 h and 24 h. We concluded that p26 might be involved in protecting the embryo from physiological stress during embryonic development.

A small heat shock protein of Ostertagia ostertagi: stage-specific expression, heat inducibility, and protection trial.

In this study, we isolated and analyzed a small heat shock protein (HSP) of Ostertagia ostertagi (Oo-HSP18). Oo-hsp18 is encoded by a single-copy gene and the full-length cDNA represents an 18-kDa protein. The expression of Oo-hsp18 is highly stage specific and restricted to the adult stage. The protein is synthesized in a tissue-specific manner and localized in the body muscle layer. The levels of Oo-hsp18 mRNAs are sharply induced by heat shock but not by other stressors such as levamisole and H2O2. A vaccination trial with recombinant Oo-HSP18 failed to protect calves against a challenge infection.